红橘响应褐斑病菌侵染的转录组学分析

doi:10.3864/j.issn.0578-1752.2020.22.006

Abstract

Abstract:

【Objective】The objective of this study is to reveal the gene expression pattern at transcriptional level of tangerine (Citrus reticulata Blanco, cv. Hongjv) after inoculated with the brown spot pathogen (Alternaria alternata tangerine pathotype), and to identify the key genes of tangerine responding to the pathogen infection.【Method】Detached leaves of tangerine were inoculated with A. alternata tangerine pathotype for 28 h and used for RNA extraction and transcriptome sequencing. Sequencing results were verified by quantitative real-time PCR (qRT-PCR). Differentially expressed genes (DEGs) were screened with |log₂fold change|≥1, q-value≤0.01 as threshold and clean reads were compared with genome of sweet orange (Citrus sinensis). GO database was used to perform functional classification of DEGs, KEGG was used to analyze metabolic pathways, and MapMan software was used to analyze the expression changes of genes related to biological stress signaling pathways.【Result】A large number of DEGs related to stress were produced at 28 h post inoculation, 5 173 up-regulated and 6 555 down-regulated genes were obtained. GO functional classification showed that the DEGs were mainly related to protein binding, membrane and oxidation-reduction process. KEGG enrichment and MapMan software analysis revealed that the basic metabolism of tangerine was severely damaged during the stress of A. alternata tangerine pathotype. The expression of multiple genes in host defense-related plant hormone signal transduction pathways such as ethylene (ET), salicylic acid (SA) and auxin (AUX) varied to some extent, of which ET plays a key role. The three members of the ET receptor ETR were activated to different degrees, among which downstream kinases and ET response factors were up-regulated, whereas most key signal genes of AUX, most members of AUX response factor ARF and genes in SA synthesis pathway were all down-regulated. Meanwhile, genes related to flavonol, anthocyanins, terpenoids and alkaloid synthesis were drastically changed by the fungus. Most genes of terpenoid synthesis were down-regulated, while the number and expression trend of up-regulated genes related to flavonoid synthesis were stronger than those of the down-regulated genes. Glucosinolate genes with anti-insect and antibacterial effects were up-regulated. Furthermore, a large number of transcription factors involved in stress resistance processes such as WRKY, bZIP, ERF, MYB and NAC were induced and activated. Most members of WRKY and bZIP transcription factors were positively regulated by the fungus, and the expressions of more than 50% of the ERF family genes were up-regulated. Under the regulation of transcription factors, PTI and ETI response genes such as receptor kinases, R proteins and NBS anti-disease proteins were largely expressed, multiple resistance PR genes were up-regulated and the expressions of 22 POD genes of antioxidant protective enzyme systems were largely activated by reactive oxygen species. The above results indicated that A. alternata tangerine pathotype infection had a great effect on the physiological state of citrus plants. To validate the findings, 19 DEGs related to anti-disease were selected for further analyses using qRT-PCR. The result was fairly consistent with that of transcriptional sequencing.【Conclusion】The DEGs and the significantly up-regulated expression genes obtained in C. reticulata Blanco, cv. Hongjv were mainly involved in the metabolic process, the response to stress and the transcriptional regulation. The synergistic regulation of these genes is an important mechanism of defense reaction to A. alternata tangerine pathotype.

Key words: Citrus reticulata Blanco, cv. Hongjv, Alternaria alternata tangerine pathotype, transcriptome, differentially expressed gene (DEG)

TANG KeZhi,ZHOU ChangYong. Transcriptome Analysis of Citrus reticulata Blanco, cv. Hongjv Infected with Alternaria alternata Tangerine Pathotype[J].Scientia Agricultura Sinica, 2020, 53(22): 4584-4600.

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References 54

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基因ID Gene ID	引物序列 Primer sequence (5′-3′)		产物长度 Length of product (bp)
Cs4g12760	F: TTCGTCTTGCTCTTCGGATAA	R: GCACTCCAACGGAATCTCTAA	202
Cs2g09310	F: GGTGTCATTCCTCCTCCTACC	R: GCAGTTCCCTCGCCTATTCT	198
Cs5g15470	F: CAGCCTTGTCGGTATGAGAA	R: CAACACCCAATCTTCCTTGAG	151
Cs5g21860	F: GCTCTTGTGGGCATTCTTGC	R: CTCTCGTGTAGAAGCTCTTGCC	142
Cs7g20700	F: ACGGTTCAGGCTCATTTCAG	R: TGGGATTTGGCATCATCAAT	185
Cs3g10430	F: TGGAGACGTAGAGGCTGTCAA	R: CATACCAATATTTTGAGCATTTT	121
Cs6g07410	F: GGAGAGTGGTGGAATGCTGAT	R: CACTTCGAGCGTGTAGGTTTG	150
Cs6g20850	F: CCAGCAGGCATGAGAAATTA	R: TGACCATCGTGGGAACAGTA	229
Cs1g01180	F: TGCAGTAGAAGTTGATGGTGATG	R: AATGAGCCGTTAGCGACAAG	165
Cs1g04680	F: AGCTGCAAGGGTTTGGTTAG	R: GAATTTGCGTTTGGTGATGA	150
Cs8g13680	F: GCCTATGCTTGCTGTTGTTTC	R: GAAGGCAGTCCATCCATACTTC	194
orange1.1t03118	F: CAAGCTCTCCAGGCAAGAGT	R: GGTCCACGGCCATAGTAGAA	247
orange1.1t03618	F: CGGGATGAACATTTGGTTTA	R: CTAGCCTTCTGATCTTGACACA	184
orange1.1t05311	F: GCTGGGATATAACTCCTTCTCA	R: TTCCGCTAAACCAATCACTT	172
Cs2g06120	F: GCACAAGGAAATGGGTTTGT	R: GAAACACGCTGGGATCACTT	229
Cs6g07400	F: ACATGGCTGCAAGAGCATAC	R: CCATTGAGGTCCACCACTTA	197
orange1.1t00214	F: ACGCTCTGTCCCTCAACAAG	R: CCGCTACTGCCTCCTGTATC	171
orange1.1t02319	F: GGGATCTACTGCCGACACTC	R: CGACGACGACCTTTGATCTT	245
orange1.1t03603	F: GGACAATGCTGATCCGAAAG	R: TCAACCAAGCCTCCTGAAAC	203
CitActin	F: CATCCCTCAGCACCTTCC	R: CCAACCTTAGCACTTCTCC	191

样本 Sample	总序列数 Total reads	比对上序列比例 Total mapped reads (%)	比对上唯一位置序列比例 Unique mapped reads (%)	双端比对上序列比例 Reads mapped in paired (%)
C0_1	47713764	90.30	86.99	84.18
C0_2	54696950	88.96	85.64	82.20
C0_3	52284118	89.93	86.59	83.36
ZC_1	55857454	75.22	71.84	69.94
ZC_2	53372222	74.18	70.98	68.37
ZC_3	57202046	74.31	70.99	68.87

Transcriptome Analysis of Citrus reticulata Blanco, cv. Hongjv Infected with Alternaria alternata Tangerine Pathotype

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